RESUMEN
Diphosphoinositol pentakisphosphate (InsP7), a higher inositol phosphate containing energetic pyrophosphate bonds, is beginning to emerge as a key cellular signaling molecule. However, the various physiological and pathological processes that involve InsP7 are not completely understood. Here we report that cigarette smoke (CS) extract and nicotine reduce InsP7 levels in aging neutrophils. This subsequently leads to suppression of Akt deactivation, a causal mediator of neutrophil spontaneous death, and delayed neutrophil death. The effect of CS extract and nicotine on neutrophil death can be suppressed by either directly inhibiting the PtdIns(3,4,5)P3/Akt pathway, or increasing InsP7 levels via overexpression of InsP6K1, an inositol hexakisphosphate (InsP6) kinase responsible for InsP7 production in neutrophils. Delayed neutrophil death contributes to the pathogenesis of CS-induced chronic obstructive pulmonary disease. Therefore, disruption of InsP6K1 augments CS-induced neutrophil accumulation and lung damage. Taken together, these results suggest that CS and nicotine delay neutrophil spontaneous death by suppressing InsP7 production and consequently blocking Akt deactivation in aging neutrophils. Modifying neutrophil death via this pathway provides a strategy and therapeutic target for the treatment of tobacco-induced chronic obstructive pulmonary disease.
Asunto(s)
Fosfatos de Inositol/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Nicotina/farmacología , Fumar , Animales , Muerte Celular , Membrana Celular/metabolismo , Separación Celular , Citometría de Flujo , Fosfatos de Inositol/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Neutrófilos/patología , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Overexpression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions. To explore chemical inhibitors against TNFα activity, we applied computer-aided drug design combined with in vitro and cell-based assays and identified a lead chemical compound, (E)-4-(2-(4-chloro-3-nitrophenyl) (named as C87 thereafter), which directly binds to TNFα, potently inhibits TNFα-induced cytotoxicity (IC50 = 8.73 µM) and effectively blocks TNFα-triggered signaling activities. Furthermore, by using a murine acute hepatitis model, we showed that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival. Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat TNFα-mediated inflammatory diseases.
Asunto(s)
Hepatitis Animal/prevención & control , Inflamación/prevención & control , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Western Blotting , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células HL-60 , Hepatitis Animal/genética , Hepatitis Animal/metabolismo , Humanos , Hidrazonas/química , Hidrazonas/farmacología , Inflamación/genética , Inflamación/metabolismo , Células Jurkat , Células K562 , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Unión Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Tiazoles/química , Tiazoles/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Background: Successful treatment of infectious bone defect remains a major challenge in the orthopaedic field. At present, the conventional treatment for infectious bone defects is surgical debridement and long-term systemic antibiotic use. It is necessary to develop a new strategy to achieve effective bone regeneration and local anti-infection for infectious bone defects. Methods: Firstly, vancomycin / poly (lactic acid-glycolic acid) sustained release microspheres (VAN/PLGA-MS) were prepared. Then, through the dual-nozzle 3D printing technology, VAN/PLGA-MS was uniformly loaded into the pores of nano-hydroxyapatite (n-HA) and polylactic acid (PLA) scaffolds printed in a certain proportion, and a composite scaffold (VAN/MS-PLA/n-HA) was designed, which can not only promote bone repair but also resist local infection. Finally, the performance of the composite scaffold was evaluated by in vivo and in vitro biological evaluation. Results: The in vitro release test of microspheres showed that the release of VAN/PLGA-MS was relatively stable from the second day, and the average daily release concentration was about 15.75 µg/mL, which was higher than the minimum concentration specified in the guidelines. The bacteriostatic test in vitro showed that VAN/PLGA-MS had obvious inhibitory effect on Staphylococcus aureus ATCC-29213. Biological evaluation of VAN/MS-PLA/n-HA scaffolds in vitro showed that it can promote the proliferation of adipose stem cells. In vivo biological evaluation showed that VAN/MS-PLA/n-HA scaffold could significantly promote bone regeneration. Conclusion: Our research shows that VAN/MS-PLA/n-HA scaffolds have satisfying biomechanical properties, effectively inhibit the growth of Staphylococcus aureus, with good biocompatibility, and effectiveness on repairing bone defects. The VAN/MS-PLA/n-HA scaffold provide the clinic with an application prospect in bone tissue engineering.
Asunto(s)
Durapatita , Vancomicina , Durapatita/farmacología , Vancomicina/farmacología , Andamios del Tejido , Microesferas , Preparaciones de Acción Retardada/farmacología , Regeneración Ósea , Poliésteres/farmacología , Impresión Tridimensional , OsteogénesisRESUMEN
Triple-negative breast cancer (TNBC) is a malignant tumor that threatens women's health. Exploring novel development-associated biomarkers could help improve the survival rate of TNBC. This study evaluated the significance and mechanism of LINC01315 in TNBC progression aiming to identify a potential biomarker. There were 103 TNBC patients that provided clinical tissues in this study. The expression of LINC01315 was assessed by PCR and its association with clinical data was evaluated by statistical analyses. The in vitro cell experiments were conducted to estimate the biological effect of LINC01315 and its molecular mechanism. A significant upregulation of LINC01315 was observed in TNBC, which was associated with disease development and severity of patients. The upregulation of LINC01315 could be a symptom of the poor prognosis of patients. The knockdown of LINC01315 suppressed the main cellular processes of TNBC progression. Additionally, miR-876-5p was demonstrated to be a target of LINC01315 and regulate the expression of GRK5, through which LINC01315 modulated the progression of TNBC. Upregulated LINC01315 in TNBC indicated the malignant development and poor survival rate of patients. Inhibition of LINC01315 might be a potential therapeutic strategy of TNBC.
Asunto(s)
Quinasa 5 del Receptor Acoplado a Proteína-G , MicroARNs , ARN Largo no Codificante , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
As a previously discovered target of DNA damage, Na+/H+ exchanger 1 (NHE1) plays a role in regulation of intracellular pH (pHi) through the extrusion of intracellular proton (H+) in exchange for extracellular sodium (Na+). Its abnormal expression and dysfunction have been reported in solid tumor and hematopoietic malignancies. Here, we reported that suppression of NHE1 in BCR-ABL+ hematopoietic malignancies' K562 cells treated with Etoposide was manipulated by miR-19 and c-MYC. Inhibition of miR-19 or c-MYC enhanced the expression of NHE1 and sensitized K562 cells to Etoposide in vitro. The in vivo nude mouse transplantation model was also performed to confirm the enhanced sensitivity of K562 cells to Etoposide by inhibiting the miR-19 or c-MYC pathway. TCGA analysis conferred a negative correlation between miR-19 level and leukemia patients' survival. Thus, our results provided a potential management by which the c-MYC-miRNA 19 pathway might have a crucial impact on sensitizing K562 cells to Etoposide in the therapeutic approaches.
Asunto(s)
Neoplasias Hematológicas , Leucemia Mielógena Crónica BCR-ABL Positiva , MicroARNs , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Animales , Etopósido/farmacología , Etopósido/uso terapéutico , Regulación Leucémica de la Expresión Génica , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) acts as a downstream effector of the receptor tyrosine kinase-Ras signaling pathway and has been reported as a candidate tumor suppressor gene in various cancers. Our current study was aimed at investigating the prognostic impact of CIAPIN1 on Non-Small-Cell Lung Carcinoma (NSCLC) patients and the effect of CIAPIN1 on NSCLC A549 cells' metastasis. METHODS: Western blot analysis was applied to detect CIAPIN1 expression; Kaplan-Meier survival analysis was used to evaluate the effect of CIAPIN1 on NSCLC patients' prognosis. Wound healing assay, Transwell chamber invasion analysis, and tumorigenicity assay in BALB/c nude mice were used to measure the metastasis potential of A549 cells. RESULTS: We found that CIAPIN1 overexpression indicated good survival duration during the follow-up period. CIAPIN1 overexpression inhibited the migration, invasion, MMPs, and EMT-associated markers in A549 cells. Further, NHE1 (Na+/H+ exchanger 1) expression and ERK1/2 phosphorylation decreased along with CIAPIN1 upregulation. Importantly, treating A549 cells with CIAPIN1 overexpression with the NHE1-specific inhibitor, Cariporide, further inhibited the metastatic capacity, MMP expression, EMT-associated markers, and phosphorylated ERK1/2. Treatment with the MEK1-specific inhibitor, PD98059, induced nearly the same suppression of CIAPIN1 overexpression-dependent metastatic capacity, MMP expression, and EMT-associated markers as was observed with Cariporide. Further, Cariporide and PD98059 exert synergistical suppression of A549 cells' metastatic capacity. CONCLUSION: Thus, the current results implied a potential management by which CIAPIN1 upregulation may have a crucial effect on the suppression of NSCLC, indicating that overexpression of CIAPIN1 might serve as a combination with chemotherapeutical agents in NSCLC therapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Pronóstico , Reproducibilidad de los Resultados , Análisis de Supervivencia , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Chronic neutrophilic leukemia (CNL) is a rare type of myeloproliferative neoplasm, characterized by sustained neutrophilia, splenomegaly, bone marrow granulocytic hyperplasia (without evidence of dysplasia) and an absence of the Philadelphia chromosome. Thus far, ~150 cases of CNL have been described in the literature; however, none have demonstrated overexpression of the ecotropic viral integration site-1 (EVI-1, also known as MECOM) gene. The present study describes a case that fulfilled the World Health Organization diagnostic criteria for CNL, and was associated with overexpression of EVI-1, as well as novel concurrent mutations of colony stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1). In addition, the current study briefly reviewed the relevant literature regarding novel genetic findings associated with the diagnosis and treatment of CNL. To the best of our knowledge, this is the first case report of CNL with associated EVI-1 overexpression, and concurrent CSF3R and SETBP1 mutations.
RESUMEN
In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions.
Asunto(s)
Apoptosis/inmunología , Quimiotaxis/inmunología , Neutrófilos/inmunología , Estallido Respiratorio/inmunología , Actinas/genética , Actinas/inmunología , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/inmunología , Quimiotaxis/efectos de los fármacos , Escherichia coli/inmunología , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Inmunidad Innata , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Transducción de SeñalRESUMEN
Anti-endothelial cell antibody (AECA) is well known to reflect endothelial injury. Graft-versus-host disease (GVHD), a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), is also closely associated with endothelial injury. We hypothesized that AECA may be associated with GVHD. To investigate the clinical significance of AECA in allo-HSCT recipients with GVHD, we detected AECA by cyto-enzyme-linked immunosorbent assay (cyto-ELISA) in allo-HSCT recipients with acute and/or chronic GVHD (aGVHD and cGVHD). Incidences of anti-HMEC-1 AECA (anti-HMEC) and anti-EA.hy926 AECA (anti-EAHY) were significantly higher in patients with grade II-IV than grade 0-I aGVHD (P = 0.049, P = 0.011, respectively). There was no difference in the incidence of AECA between patients with and without cGVHD. Patients with anti-EAHY positive in the early stage post-transplant demonstrated a higher incidence of cGVHD (P = 0.044). In patients with grade 0-I aGVHD, AECA-positive patients had higher overall survival and disease-free survival (P < 0.05), and tended to have lower incidences of relapse and transplant-related mortality. Our data suggest that AECA plays an important role in the pathogenesis of GVHD.
Asunto(s)
Autoanticuerpos/sangre , Células Endoteliales/inmunología , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Aloinjertos , Autoanticuerpos/fisiología , Biomarcadores/sangre , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Tasa de Supervivencia , Venas Umbilicales/citología , Adulto JovenRESUMEN
Caspase-3-mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8- or caspase-9-mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death.
Asunto(s)
Caspasa 3/metabolismo , Inflamación , Mieloblastina/metabolismo , Neutrófilos/patología , Animales , Apoptosis , Células de la Médula Ósea/citología , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Separación Celular , Modelos Animales de Enfermedad , Activación Enzimática , Citometría de Flujo , Humanos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/citología , Neutrófilos/metabolismo , Peritonitis/metabolismo , SuperóxidosRESUMEN
AIM: To prepare functional monoclonal antibodies(mAb)against recombinant human Flt-1(rhFlt-1). METHODS: A cell line stable secreting mAb was established by using FLT-1 extracellular domain III as antigen and hybridoma technique. Then it was purified in large-scale from mouse ascites by protein G affinity chromatography. The characteristics of mAb were then determined by ELISA, Western blotting and FACS. RESULTS: The immunoglobin subtype of mAb XA12 was IgG1 with kappa (κ) light chains, and it could recognize rhFlt-1 specifically. Furthermore, mAb XA12 could bind to rhFlt-1with high affinity (K=1.28±0.05 nmol/L). It could also be used to detect Flt-1-positive cells, such as human umbilical vascular endothelial cells (HUVECs) and K562/A02 in a dose-dependent fashion. CONCLUSION: A hybridoma cell line secreting functional anti-rhFlt-1 mAb was successfully prepared. The antibody can be used to study the function of Flt-1 and further potentially optimized for clinical purpose.